Fig 2: MCAM is highly expressed in AM cell lines. (A) Scatter plot of DNA microarray comparing NHEM and SM2‐1 (left) or NHEM and MMG‐1 (right). Red dots indicate genes upregulated in AM cell lines with Z‐score ≥ 2.0 and ratio ≥ 2.0. Blue dot indicates MCAM. (B and C) Expression of MCAM mRNA (B) and MCAM protein (C) in NHEK, NHEM, SM2‐1, and MMG‐1 cells. Mean ± SD of the expression of MCAM calculated from three independent experiments and representative blot images are shown. Original, uncropped blot images are shown in Figure S2. **p < 0.01 and ***p < 0.001. (D) Representative immunocytochemical images of MCAM in NHEK, NHEM, SM2‐1, and MMG‐1 cells. Scale bars = 100 μm.

Fig 3: Effect of MCAM knockdown on proliferation of AM cell lines. NHEK, NHEM, SM2‐1, and MMG‐1 cells were transfected with control or MCAM siRNA and their proliferation was evaluated. (A) Fold change of viable cells under the control or MCAM siRNA‐transfected conditions. Experiments were performed in three wells per condition and independently repeated three times. **p < 0.01 and ***p < 0.001. (B) Expression of CCND1, BCL2, and BAX mRNA in control or MCAM siRNA‐transfected cells. Mean ± SD of the expression of each gene calculated from three independent experiments is shown. *p < 0.05, **p < 0.01, and ***p < 0.001. (C and D) Expression of cyclin D1, BCL2, and BAX proteins in control or MCAM siRNA‐transfected cells. Representative blot images (C) and mean ± SD of each protein expression (D) calculated from three independent experiments are shown. Original, uncropped blot images are shown in Figure S7. N.D., Not detected. *p < 0.05 and ***p < 0.001.

Fig 4: Effect of MCAM knockdown on migration of AM cell lines. NHEK, NHEM, SM2‐1, and MMG‐1 cells were transfected with control or MCAM siRNA and their migration was evaluated. (A) Expression of CDH1 and VIM mRNA in control or MCAM siRNA‐transfected cells. Mean ± SD of the expression of each gene calculated from three independent experiments is shown. **p < 0.01 and ***p < 0.001. (B and C) Expression of E‐cadherin and vimentin proteins in control or MCAM siRNA‐transfected cells. Representative blot images (B) and mean ± SD of the expression of each protein (C) calculated from three independent experiments are shown. Original, uncropped blot images are shown in Figure S8. *p < 0.05 and **p < 0.01. (D) Cell migration assay of control or MCAM siRNA‐transfected SM2‐1 and MMG‐1 cells in the presence or absence of mitomycin C (MMC, final concentration of 1.25 μg/mL). Mean ± SD of the relative wound area calculated from three independent experiments with six replicate wells per condition are shown.